Agent for applying to mucosa and method for the production thereof

ABSTRACT

An agent for applying to mucosa capable of persistently exerting a therapeutic effect on disorders such as inflammation and lesions in the mucosa even by a lower frequency of administration because the agent can stay at a diseased site for a long period of time by exhibiting a high staying properly in a mucosal epithelial layer is provided, said agent for application to mucosa containing glycosaminoglycan (GAG) into which a hydrophobic group is introduced via a binding chain, as an active ingredient.

TECHNICAL FIELD

The present invention relates to an agent for applying to mucosacontaining a hydrophobic group binding type glycosaminoglycan as anactive ingredient, and a method for production thereof.

BACKGROUND ART

Conventionally, as substances having healing effects on mucosaldisorders such as inflammation and lesions, hyaluronic acid which is arepresentative glycosaminoglycan (hereinafter described as “GAG”) hasbeen known (e.g., Patent Document 1). However, in mucosa in the cornea,oral cavity and nasal cavity, and conjunctiva which contacts with theexternal world, and mucosa in the urinary bladder, the mucosal surfacesare washed with secretions and excretions such as tear fluid, salivaryfluid and urine, and foreign substances are removed. Thus derivatives ofGAG which keep medicinal effects inherent in GAG and exert a highstaying property in these mucosal tissues have been demanded.

Meanwhile, photoreactive hyaluronic acid whose water-solubility has beenincreased by binding a photocrosslinking group such as cinnamic acid tohyaluronic acid and further giving an alkali treatment thereto has beenknown (e.g., Patent Document 2). This photoreactive hyaluronic acid hasbeen provided by binding a photocrosslinking group such as cinnamic acidto hyaluronic acid in order to provide medical materials such asanti-adhesion materials by giving the photocrosslinking, and does notaim at enhancing the staying property in the mucosal tissue.

[Patent Document 1] Japanese Published Unexamined Patent Publication No.Hei-1-238530

[Patent Document 2] Japanese Published Unexamined Patent Publication No.2002-249501

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

The present invention aims at providing an agent for applying to mucosa,which exerts an excellent staying property and pharmacological effectsin mucosa.

Means for Solving the Problem

As a result of an extensive study for solving the above problem, thepresent inventors have found that “hydrophobic group binding type GAG”obtained by binding a hydrophobic group to GAG via a binding chain canbe used as an extremely excellent active ingredient in an agent forapplying to mucosa because this GAG keeps healing effects inherent inGAG on mucosal disorders such as inflammation and lesions and exhibits ahigh staying property when applied thereto, and have completed thepresent invention.

The present invention relates to an agent for applying to mucosa whichcontains glycosaminoglycan (GAG) into which the hydrophobic group isintroduced via the binding chain.

Effects of the Invention

The agent for applying to mucosa of the present invention can exert thepersistent healing effect on the mucosal disorder such as inflammationand lesions even by low frequent administration because this can stay ata diseased site for a long period of time by exhibiting high stayingproperty in the mucosa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing a spectrum of light transmittance;

FIG. 2 is a view showing healed area percentages;

FIG. 3 is a view showing healed areas;

FIG. 4 is a view showing healing rate;

FIG. 5 is a view showing healed areas;

FIG. 6 is a view showing healing rate;

FIG. 7 is a view showing healed areas;

FIG. 8 is a view showing healing rate;

FIG. 9 is a view showing healed areas;

FIG. 10 is a view showing healing rate;

FIG. 11 is a, view showing staying property at peeled sites in rabbitcorneal epithelia;

FIG. 12 is a view showing staying property at peeled sites in rabbitcorneal epithelia;

FIG. 13 is a view showing photographs of an eyeball after irradiationwith ultraviolet rays;

FIG. 14 is a view showing amounts of water evaporation in a removedcornea;

FIG. 15 is a view showing amounts of water evaporation in a removedcornea; and

FIG. 16 is a view showing changes in water evaporation amount ratio inmodel hamsters for xerostomia.

FIG. 17 is a view showing healed areas;

FIG. 18 is a view showing healing rate;

FIG. 19 is a view showing healed areas;

FIG. 20 is a view showing healing rate.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below by the bestmodes for carrying out the invention.

Herein, an alkyl group refers to a straight or branched aliphatichydrocarbon group having. a described number of carbon atoms. An alkenylgroup refers to a straight or branched aliphatic hydrocarbon grouphaving a described number of carbon atoms, having at least one doublebond. An alkynyl group refers to a straight or branched aliphatichydrocarbon group having a described number of carbon atoms, having atleast one triple bond.

An aryl group refers to a monocyclic or polycyclic aromatic hydrocarbongroup having 6 to 20 carbon atoms as ring-constituting atoms. Aheteroaryl group refers to a monocyclic or polycyclic aromatichydrocarbon group having 3 to 20 carbon atoms and one or heteroatomsselected from nitrogen, sulfur and oxygen atoms as the ring-constitutingatoms.

An arylalkyl group refers to the alkyl group defined above substitutedwith the aryl group defined above. An arylalkynyl group refers to thealkenyl group defined above substituted with the aryl defined above. Anarylalkynyl group is the alkynyl group defined above substituted withthe aryl group defined above.

An amino acid group refers to a group derived by losing a carboxylgroup, an amino group or a hydroxyl group by a chemical bond from anatural or synthetic amino acid.

Herein, the term “treatment” includes prevention, control of progression(prevention of deterioration), improvement (reduction) and cure of themucosal disorder. The “mucosal disorder” means a condition wheremorphology, properties and functions to be inherent in the mucosa aredisordered in some form. For example, the mucosal disorder can includethe conditions such as lesions, defects, erosion, inflammation, ulcersand dryness.

GAG into which the hydrophobic group is introduced via the bindingchain, which is contained as the active ingredient in the agent forapplying to mucosa of the present invention can be any GAG as long asthe GAG binds the group having hydrophobicity derived from a hydrophobiccompound having a water, insoluble and oil soluble nature. Thishydrophobic group is bound to GAG via the binding chain. As describedlater, it is not necessary that all constitutive units of GAG bind thehydrophobic groups.

1) GAG

GAG in GAG into which the hydrophobic group is introduced via thebinding chain contained in the agent for applying to mucosa of thepresent invention is an acidic polysaccharide having a repeated longchain structure of disaccharide composed of amino sugar and uronic acid(or galactose). Examples of such GAG include hyaluronic acid,chondroitin, chondroitin sulfate, heparin, heparan sulfate, dermatansulfate and keratan sulfate, and among them, hyaluronic acid ispreferable. These GAG may be pharmaceutically acceptable salts thereof.Examples of such salts include sodium salts, potassium salts, magnesiumsalts and calcium salts, and among them, the sodium salt is preferable.Therefore, it is the most preferable that GAG in the agent for applyingto mucosa of the present invention is sodium hyaluronate. An origin ofGAG is not particularly limited, and GAG may be derived from an animalor a microorganism or chemically synthesized. For example, when usingsodium hyaluronate, those derived from cock's comb can be exemplified.The molecular weight of GAG is not particularly limited, but its weightaverage molecular weight is preferably 200,000 to 3,000,000, morepreferably 500,000 to 2,000,000 and most preferably 600,000 to1,200,000. When hyaluronic acid or the pharmaceutically acceptable saltthereof is used, its weight average molecular weight is preferably200,000 to 3,000,000, more preferably 500,000 to 2,000,000 and mostpreferably 600,000 to 1,200,000.

2) Hydrophobic Group

The hydrophobic group in GAG into which the hydrophobic group isintroduced via the binding chain contained in the agent for applying tomucosa of the present invention is any group as long as the hydrophobicgroup is derived from the compound having the water insoluble and oilsoluble nature. Examples of such a group can include alkyl groups having2 to 18 carbon atoms, alkenyl groups having 2 to 18 carbon atoms,alkynyl groups 15 having 2 to 18 carbon atoms, aryl groups, heteroarylgroups, arylalkyl groups, arylalkenyl groups, arylalkynyl groups andamino acid groups.

The alkyl groups having 2 to 18 carbon atoms can include methyl, ethyl,n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl,isopentyl, neopentyl, n-heptyl, 5-methylhexyl, 4,4-dimethyl-pentyl,1,1-dimethyl-pentyl and n-octyl. Among them, the alkyl groups such asn-butyl having 2 to 6 carbon atoms such as n-butyl can be preferablyincluded.

The alkenyl groups having 2 to 18 carbon atoms can include vinyl,1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl,1-methyl-1-propenyl, 1-pentenyl, 3-methyl-2-butenyl, 1-heptene-1-yl and2-heptene-1-yl. Among them, the alkenyl groups such as 1-butenyl having2 to 6 carbon atoms such as 1-butenyl can be preferably included.

The alkynyl groups having 2 to 18 carbon atoms can include ethynyl,1-propinyl, 2-propinyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-heptynyl,2-heptynyl and 3-heptynyl. Among them, the alkynyl groups such as1-butynyl having 2 to 6 carbon atoms such as 1-butynyl can be preferablyincluded.

The aryl groups can include groups such as phenyl, naphthyl, anthryl andphenanthryl.

The heteroaryl groups can include groups such as furyl, thionyl,thiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidinyl and indolyl.

The arylalkyl groups can include groups such as benzyl, phenethyl,naphthylmethyl and naphthylethyl.

The arylalkenyl groups can include groups such as 2-phenyl-ethenyl andp-aminophenylethenyl.

The arylalkynyl groups can include groups such as 2-phenyl-ethynyl andp-aminophenylethynyl.

The amino acid groups can include groups derived from aliphatic aminoacids such as glycine, alanine and 3-alanine; branched aliphatic aminoacids such as leucine, isoleucine and valine; aromatic amino acids suchas phenylalanine and tyrosine; and heterocyclic amino acids such astryptophan and histidine.

These hydrophobic groups may be monosubstituted or polysubstituted withgroups such as hydroxyl, carboxyl, cyano, amino (which may bemonosubstituted or disubstituted with the above alkyl), nitro, oxo andalkylcarbonyloxy.

Among the above hydrophobic groups, aryl groups, arylalkyl groups,arylalkenyl groups and arylalkynyl groups which are the hydrophobicgroups containing the aryl group can be preferably included, and thearylalkenyl group and the aryl group substituted with thealkylcarbonyloxy group can be particularly preferably included. As suchan arylalkenyl group, it is possible to specifically use phenylethenyland p-aminophenylethenyl. As the aryl group, it is possible topreferably use the groups such as CH3-(CH01-000-phenyl (wherein 1represents or an integer of 1 to 18).

These hydrophobic groups may also have a function such as ultravioletray absorption ability due to having a double bond in the hydrophobicgroup as shown by a functional group such as phenyl-ethenyl exemplifiedabove contained in the hydrophobic group. For example, when the agentfor applying to mucosa of the present invention is used as eye dropsdescribed later, it is possible to make the eye drops having thefunction effectively absorbing the harmful ultraviolet rays by the useof the group having the ultraviolet ray absorption ability as thehydrophobic group. Furthermore, for example, when the agent for applyingto mucosa of the present invention is used for the treatment of cornealepithelial layer disorders such as corneal xerosis (dry eye),keratoconjunctivitis, superficial punctate keratitis (SFK), cornealepithelial erosion, corneal epithelial loss and corneal tumor, it ispossible to make the agent for applying to mucosa which haspharmacological effects on the above disorders in combination with thefunction effectively absorbing the harmful ultraviolet rays by the useof the group having the ultraviolet ray absorption ability as thehydrophobic group. As the group having the ultraviolet ray absorptionability, for example, arylalkenyl group having the conjugation doublebond which is exemplified by 2-phenyl-ethenyl and p-aminophenylethenyldescribed above is preferable. When the agent for applying to mucosa ofthe present invention is used for the corneal disorder, it is preferablethat “GAG into which the hydrophobic group is introduced” which is theactive ingredient of the agent for applying to mucosa of the presentinvention is made into an aqueous solution of 0.1% by weight, whichblocks 70 to 100% transmission of the ultraviolet rays at a wavelengthof 200 to 300 nm when an ultraviolet ray transmittance is measured bythe method described in the Example described later. Such a hydrophobicgroup having the ultraviolet ray absorption ability can preferablyinclude the arylalkenyl groups such as 2-phenyl-ethenyl andp-aminophenylethenyl.

3) Binding Chain

In GAG into which the hydrophobic group is introduced via the bindingchain contained in the agent for applying to mucosa of the presentinvention, the above GAG is bound to the above hydrophobic group via thebinding chain. GAG has the functional group which is a carboxyl,hydroxyl or sulfonate (—S0₃H) group as the side chain. Thus, thehydrophobic group can be bound to GAG via the binding chain obtained byforming an ether bond, carboxylate ester bond, sulfate ester bond,carboxylic acid amide bond or sulfonate amide bond together with thesefunctional groups. Such a binding chain can specifically include —CONH—,—COO—, —O—, —SO₃,— and —SO₂NH—. Among them, the carboxylic acid amidebond of —CONH— and the carboxylate ester bond of —COO— can be preferablyused, and the carboxylic acid amide bond of —CONH— can be particularlypreferably used.

4) Spacer Chain

In GAG into which the hydrophobic group is introduced via the bindingchain contained in the agent for applying to mucosa of the presentinvention, the hydrophobic group is bound to GAG via the above bindingchain, and a spacer chain may further exist between the binding chainand the hydrophobic group. As such a spacer chain, any chain group canbe used as long as the spacer group does not completely lose thepharmacological effects which GAG has. Specifically, —(CH₂)_(m)— and—(CH₂)—(OCH₂)_(n)— (wherein m and n are integers of 1-to 18,respectively) can be included.

These spacer chains can further have the binding chains such as —CONH—,—COO—, —O—, —SO₃— and —SO₂NH— which are the same as above at thehydrophobic group side. Such a spacer chain having the binding chain atthe hydrophobic group side can specifically include —COO—(CH₂)_(m)—,—COO—CH₂)—(OCH₂)_(n)-1, —CONH—(CH₂)_(m)— and —CONH—(CH₂)—(OCH₂)_(n)—.

5) Ratio Having Hydrophobic Group

In GAG into which the hydrophobic group is introduced via the bindingchain contained in the agent for applying to mucosa of the presentinvention, it is not necessary that all of GAG constitutive unitsrespectively have the hydrophobic groups. A ratio of, the boundhydrophobic group in molar equivalent relative to a disaccharide repeatunit in molar equivalent of GAG (hereinafter, referred to as an“introduction ratio”) can be optionally determined depending on the typeof the hydrophobic group, the degree of required hydrophobicity, thetype of the mucosal disorder administered with the agent for applying tomucosa and the administration site, etc. For example, when using aphenylethenyl group which may be substituted as the hydrophobic group,preferably 5 to 30% and more preferably 10 to 20% of a hydrophobic groupin molar equivalent is introduced relative to the disaccharide repeatunit in molar equivalent of GAG (in the case where the crosslinking bonddescribed later is not formed).

6) Crosslink Forming Group

In GAG into which the hydrophobic group is introduced via the bindingchain contained in the agent for applying to mucosa of the presentinvention, the hydrophobic group may form a crosslinking bond betweenGAG molecules by the functional group contained in the group. As thehydrophobic group capable of forming the crosslinking bond, any groupcan be used as long as the hydrophobic group produces aphotodimerization reaction or a photopolymerization reaction byirradiation of ultraviolet rays and is the same as defined above. Thehydrophobic group capable of forming the crosslinking bond includes, forexample, phenylethenyl, p-aminophenylethenyl, ethenyl, 2-carboxyethenyland pentane-1,3-dienyl. It is desirable that these groups are bound toGAG via the binding chain which contains the carbonyl group. Among thesehydrophobic groups, phenylethenyl or, p-aminophenylethenyl which isbound to GAG via the binding chain which contains the carbonyl group canbe particularly preferably used.

In such GAG into which the hydrophobic group is introduced capable offorming the crosslinking bond, GAG molecules can be crosslinked with oneanother by being subjected to the photodimerization reaction or thephotopolymerization reaction by standard methods. For example, accordingto the methods described in Japanese Published Unexamined PatentPublication No. 2002-249501, the photodimerization reaction or thephotopolymerization reaction can be given.

7) Preferable GAG Constitutive Unit

Representatives of GAG into which the hydrophobic group is introducedvia the binding chain contained in the agent for applying to mucosa ofthe present invention can specifically include the agents for applyingto mucosa containing GAG into which Ph-CH═CH—COO—(CH₂)_(m)—NH—;Ph-CH═CH—COO—CH₂—(OCH₂)_(n)—NH—; Ph-CH═CH—CONH—(CH₂)_(m)—NHCO—;Ph-CH═CH—CONH—CH₂—(OCH₂)_(n)—NHCO—; Ph-CH═CH—COO—(CH₂)_(m);Ph-CH═CH—COO—CH₂—(OCH₂)_(n)—O; Ph-CH═CH—CONH—(CH₂)_(m)—O;Ph-CH═CH—CONH—CH₂—(OCH₂)_(n)—O; CH₃—(CH₂)₁—COO-Ph-CONH—(CH₂)_(m)—NH orCH₃—(CH₂)₁—COO-Ph-CONH—CH₂—(OCH₂)n-NH (wherein Ph represents phenylgroup, m and n represent integers of 1 to 18, respectively, and 1represents 0 or an integer of 1 to 18) is introduced, as the activeingredient.

The following GAG can be included as the representative.

GAG having the repeat unit of the structural unit represented by theChemical formula 1, as a basic skeleton:

wherein, R represents R₁ or R₂;Ac represents an acetyl group;R₁ represents ONa or OH;R₂ represents (1) Ph-CH═CH—COO—(CH₂)_(n)—NH—;(2) Ph-CH═CH—COO—CH₂—(OCH₂)_(n)—NH—;(3) Ph-CH═CH—CONH—(CH₂)_(m)—NH;

(4) Ph-CH═CH—CONH—CH₂—(OCH₂), —NH—;

(5) Ph-CH═CH—COO—(CH₂)_(m), —O—;(6) Ph-CH═CH—COO—CH₂—(OCH₂)_(n)—O—;(7) Ph-CH═CH—CONH—(CH₂)_(m)—O—;(8) Ph-CH═CH—CONH—CH₂—(OCH₂)_(n)—O—;(9) CH₃—(CH₂)₁—COO-Ph-CONH—(CH₂)_(m)—NH; or(10) CH₃—(CH₂), —COO-Ph-CONH—CH₂—(OCH₂)_(n)—NH—;wherein, Ph represents phenyl group, m and n represent integers of 1 to18, respectively, and 1 represents 0 or an integer of 1 to 18, as abasic skeleton, wherein the ratio of the above structural unit wherein Rrepresents R₂ is 5 to 30% in molar equivalent relative to thedisaccharide repeat unit in molar equivalent of the GAG.8) Method for Producing GAG into which Hydrophobic Group is IntroducedVia Binding Chain

To obtain GAG into which the hydrophobic group is introduced via thebinding chain, GAG is reacted with a hydrophobic compound in which theabove hydrophobic group has been bound to the functional group such ashydroxyl, carboxyl, amino or sulfonate group which can form an etherbond, carboxylate ester bond, sulfate ester bond, carboxylic acid amidebond or sulfonate amide bond together with the carboxyl, hydroxyl orsulfonate (—S0₃H) group in GAG. Specifically, when the bond is thecarboxylic acid amide bond, GAG having the carboxyl group is reactedwith the hydrophobic compound having an amino group to bind the carboxylgroup in GAG to the amino group in the hydrophobic compound. In the caseof the carboxylate ester bond, GAG is reacted with the hydrophobiccompound having hydroxyl or carboxyl group to bind the carboxyl group inGAG to the hydroxyl group in the hydrophobic compound or bind thehydroxyl group in GAG to the carboxyl group in the hydrophobic compound.In the case of the ether bond, GAG having the hydroxyl group is reactedwith the hydrophobic compound having the hydroxyl group to react thehydroxyl group in GAG with the hydroxyl group in the hydrophobiccompound. In the case of the sulfonate ester bond, GAG is reacted withthe hydrophobic compound having the hydroxyl group or sulfonate group tobind the hydroxyl group in GAG to the sulfonate group in the hydrophobiccompound or bind the sulfonate group in GAG to the hydroxyl group in thehydrophobic compound. These reactions can be performed by commonstandard methods, and reaction conditions can be optionally selected bythose skilled in the art.

When the spacer chain is present between the binding chain and thehydrophobic group, the order in introducing the spacer chain and thehydrophobic group to GAG is not particularly limited. For example,either the method in which a spacer compound having the functional groupsuch as the hydroxyl, carboxyl, amino or sulfonate group, which can formthe ether bond, carboxylate ester bond, sulfate ester bond, carboxylicacid amide bond or sulfonate amide bond together with the functionalgroup in GAG at one end of the above spacer chain is reacted with GAG,and subsequently, the other end of the spacer compound is reacted withthe hydrophobic compound which is bound to the functional group such asthe hydroxyl, carboxyl, amino, or sultanate group, or the method inwhich the spacer compound having the functional group such as thehydroxyl, carboxyl, amino or sultanate group which can form an etherbond, carboxylate ester bond; sulfate ester bond, carboxylic acid amidebond or sulfonate amide bond together with the functional group in thehydrophobic compound at one end is reacted with the hydrophobic compoundin which the hydrophobic group has been bound to the functional groupsuch as the hydroxyl, carboxyl, amino or sulfonate group, andsubsequently the other end of the spacer compound is reacted with GAGmay be used. In particular, the method in which the spacer compound isreacted with the hydrophobic compound followed by being reacted with GAGcan be preferably used.

The above-described method can be appropriately carried out by publiclyknown methods, and preferably performed in the presence of a condensingagent. Such a condensing agent can preferably include water solublecarbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDCI.HCl), condensing agents such asdicyclohexylcarbodiimide (DCC) and N-hydroxysuccinate imide (HOSu). Forexample, when hyaluronic acid is used as GAG and the cinnamatederivative such as Ph-CH═CH—COO—(CH₂)_(m)—NH₂ orPh-CH═CH—COO—CH₂—(OCH₂)_(n)—NH₂ (wherein m and n are integers of 1 to18,respectively) is used as the hydrophobic compound which is bound tothe spacer compound, the condensation method using water solublecarbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride(EDCI.HCl) and N-hydroxysuccinate imide can be preferablyused. The reaction can be accomplished using a mixed solvent of waterand a water soluble organic solvent such as dioxane, dimethylformamideor ethanol. The hyaluronate derivative which is highly soluble in anaqueous vehicle can be obtained by treating with a base such as Sodiumhydrogen carbonate after the completion of the reaction.

When the thus produced GAG into which the hydrophobic group isintroduced via the binding chain is subjected to the photodimerizationreaction or the photopolymerization reaction to cross link the GAGmolecules with one another, for example, the method described inJapanese Published Unexamined Patent Publication No. 2002-249501 can beused. Specifically, in the case of the compound in which thephenylethenyl group as the hydrophobic group is bound to GAG via—COO—(CH₂)_(m)—NHCO—, the crosslink can be formed by irradiating light,to the solution containing them using an ultraviolet lamp.

9) Agent for Applying to Mucosa of the Present Invention

The agent for applying to mucosa of the present invention contains oneor more GAG into which the hydrophobic group is introduced via thebinding chain as the active ingredient, and may also further includeother medically, pharmaceutically or biologically acceptable substancesother than the GAG into which the hydrophobic group is introduced viathe binding chain. Such substances include but are not limited to, saltssuch as sodium chloride, potassium chloride, disodium hydrogenphosphate, sodium dihydrogen phosphate and monopotassium hydrogenphosphate, and preservatives such as paraoxybenzoate esters,benzalkonium chloride; chlorobutanol and chlorhexidine glucoriate, andother pharmacologically active ingredients.

The agent for applying to mucosa of the present invention can be madeinto any publicly known formulation. forms (e.g., solid preparationssuch as granules and powder, liquid preparations such as aqueoussolutions, suspension and emulsion, and gel preparations) as thepharmaceutical for applying to the mucosa. In the agent for applying tomucosa of the present invention, the form thereof upon formulating anddistributing and the form thereof upon applying to the mucosa may be thesame or different. For example, the agent for applying to mucosa of thepresent invention may be formulated in the form of solution and may beapplied directly to the mucosa as it is. Also, the agent for applying tomucosa of the present invention may be formulated and distributed in thesolid form, and may be made into solution or gel when being applied tothe mucosa. Thus, the agent for applying to mucosa of the presentinvention can be made into the formulation form for being prepared whenused.

When being made into the liquid agent by dissolving in water, the amountof GAG into which the hydrophobic group is introduced via the bindingchain is preferably 0.02 to 5% by weight, more preferably 0.1 to 3% byweight, extremely preferably 0.1 to 1% by weight and most preferably 0.1to 0.6% by weight.

<Applied Subjects>

The agent for applying to mucosa of the present invention aims atapplying to the mucosa. Animals to which the agent for applying tomucosa of the present invention is applied are not particularly limitedas long as they have the mucosa, and mammalian animals are preferable.The mammalian animals include, but are not limited to, humans, horses,cattle, dogs, cats, rabbits, hamsters, guinea pigs and mice. The agentfor applying to mucosa of the present invention may be of course madeinto the pharmaceuticals for humans, and can also be made into thepharmaceuticals for the animals. Among them, it is preferable to be madeinto the pharmaceuticals for humans.

The mucosa to which the agent for applying to mucosa of the presentinvention can be applied is not particularly limited as long as themucosa is the mucosa present in the animal. Such mucosa include mucosaltissues present in organs and tissues exemplified by thegastrointestinal system such as the stomach and intestines, thecardiovascular system, the respiratory system, the excretion system suchas the urinary bladder, rectum and anus, the genital system such as thevagina, and organs such as eyes, nose and oral cavity which contact withthe external world. Among them, the agent for applying to mucosa of the,present invention can be preferably applied to the cornea, conjunctiva,oral cavity mucosa and urinary bladder mucosa.

<Applied Diseases>

The agent for applying to Mucosa of the present invention can be widelyapplied to such mucosa. The purpose of the application is notparticularly limited, and for example, the purposes such as protectionof the mucosal tissue (e.g., prevention of snow blindness by ultravioletrays, pterygium and cataract), prevention of mucosal dryness and thetreatment of mucosal disorder Can be exemplified. Thus, the agent forapplying to mucosa of the present invention can be applied to not onlythe mucosa in the abnormal state (e.g., mucosa where the disorder hasoccurred) but also the mucosa in the normal state. However, since theagent for applying to mucosa of the present invention exerts excellentpharmacological effects in the mucosa where the disorder has occurred,it is possible to preferably use for the treatment of the mucosaldisorders, e.g., disorders in the cornea, conjunctiva, oral cavitymucosa and urinary bladder mucosa.

Since the agent for applying to mucosa, of the present invention exertsexcellent pharmacological effects particularly on disorders in themucosal epithelia among the mucosal disorders, it is possible to bepreferably used for the treatment of the disorders in the mucosalepithelia.

Examples of such disorders in the mucosal epithelia include cornealepithelial layer disorders such as corneal xerosis (dry eye);keratoconjunctivitis, superficial punctate keratitis (SPK), cornealepithelial erosion corneal epithelial loss and corneal tumor; oralcavity' mucosal disorders such as xerostomia (dry mouth), aphthousulcer, stomatitis and glossitis; dryness and, pruritus of nasal mucosa;urinary bladder mucosal disorders such as interstitial cystitis;ulcerative proctitis, and dryness of the rectum or vagina. Also drynessand lesions of organ mucosa upon surgical operation can be exemplified.Among them, it is, possible to be preferably used for the treatment ofthe corneal epithelial layer disorders, the oral cavity mucosalepithelial disorders and the urinary bladder mucosa epithelial layerdisorders.

Application Method and Amount

The agent for applying to mucosa of the present invention can be appliedto the mucosal tissues exemplified above, and its application method andapplication formulation can be appropriately determined by those skilledin the art depending on the position, morphology, property and functionof the mucosa to be applied, and the purpose of the application.However, it is preferable that the agent for applying to mucosa of thepresent invention is applied to the mucosa in the liquid form such assolution in use. In that case, upon producing (formulating) or applyingthe agent for applying to mucosa of the present invention, the liquidcan be obtained by dissolving GAG into which the hydrophobic group isintroduced via the binding chain in the solvent. The solvent is notparticularly limited as long as the solvent can dissolve the GAG intowhich the hydrophobic group is introduced via the binding chain and isthe pharmaceutically acceptable solvent. For example, a buffer such as aphosphate buffer or saline can be used, but the solvent is not limitedthereto. In this case, the concentration of the GAG into which thehydrophobic group is introduced via the binding chain in the liquidagent is not particularly limited, and can be appropriately determineddepending on the type of the mucosa to be applied and the degree of themucosal disorder. When the agent for applying to mucosa of the presentinvention is the eye drops, when the agent for applying to mucosa of thepresent invention is applied to the oral cavity mucosa or the urinarybladder mucosa, for example, the concentration is preferably 0.02 to 5%by weight, more preferably 0.1 to 3% by weight, still more preferably0.1 to 1% by, weight, still more preferably 0.1 to 0.6% by weight,extremely preferably 0.1 to 0.5% by weight and most preferably 0.1 to0.3% by weight.

When the agent for applying to mucosa of the present invention isapplied to the mucosa in the stomach as the liquid as above, an oraladministration or the administration using a catheter can be selected.When applied to the mucosa in the eye, the nose or the oral cavity, forexample, she administration method such as instillation of drop, nasalinstillation or oral inclusion can be selected. For example, when theagent for applying to mucosa of the present invention is applied to themucosa mucosa in the urinary bladder, rectum or vagina, or the mucosa oforgans where the dryness is concerned upon surgical operation, themethod of administering by injecting, spraying or applying the agent forapplying to mucosa of the present invention to a lumen or a surface ofthese organs or tissues can be selected, but the methods are not limitedthereto.

The amount, the number of times and the frequency of the application,(administration of) of the agent for applying to mucosa of the presentinvention is not particularly limited, and should be determineddepending on the mucosa subjected, to the application, the purpose ofthe application, the, type, age, body weight, gender, and degree ofmucosal disorder in the animal to be applied.

Specifically, when the agent for applying to mucosa of the presentinvention is used for the purpose of treating the human cornealepithelial layer disorder, the agent for applying to mucosa of thepresent invention at the above-described concentration as the liquidformulation for the instillation of drops (eye drops) containing GAGinto which the hydrophobic group is introduced via the binding chain canbe administered by instilling 1 to 3 drops per administration 1 to 5times per day, and may be administered by instilling 1 to 3 drops peradministration 1 to 3 times per day.

When the agent for applying to mucosa of the present invention is usedfor the purpose of treating the human oral cavity mucosal disorder, theagent for applying to mucosa of the present invention at theabove-described concentration as the liquid containing GAG into whichthe hydrophobic group is introduced via the binding chain can beadministered by putting the agent for applying to mucosa of the presentinvention in the oral cavity 1 to 5 times per day and rinsing forapproximately several tens of seconds (preferably approximately 20 to 30seconds) followed by spitting it out.

When the agent for applying to mucosa of the present invention isapplied to the urinary bladder mucosal disorder, this is preferably usedfor the treatment of the urinary bladder mucosal disorders exemplifiedby non-bacterial refractory cystitis exemplified by interstitialcystitis, eosinophilic cystitis and hemorrhagic cystitis which do notrespond to anti-bacterial agents although symptoms similar to those ofacute bacterial cystitis are exhibited. In this case, the agent forapplying to mucosa of the present invention at the above-describedconcentration as the liquid containing GAG into which the hydrophobicgroup is introduced via the binding chain can be administered byadministering the agent for applying to mucosa of the present inventiondirectly to the urinary bladder at the amount of 50 mL peradministration 1 to 7 times per week or administering with a catheter inthe urinary bladder.

The agent for applying to mucosa of the present invention can stay atthe diseased site for a longer period of time because the activeingredient contained in the agent exhibits the high staying property inthe mucosa, compared with the conventional drugs containing hyaluronicacid as the active ingredient in which no hydrophobic group has beenbound. Therefore, the agent for applying to mucosa of the presentinvention can also exert the treating effect persistently even at thelow administration frequency on the disorders such as inflammation andlesions in the mucosa. However, the agent for applying to mucosa of thepresent invention is not limited by its administration frequency.

The present invention will be described below by Examples.

Example 1 (1-1) Preparation of Cinnamate Derivative-Introduced SodiumHyaluronate

A 172 mg/5 mL aqueous solution of N-hydroxysuccinimide (HOSu: WatanabeChemical Industries, Ltd.), a 143 mg/5 mL aqueous solution of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI.HCl)(Watanabe Chemical Industries, Ltd.), and a 181 mg/5 mL aqueous solutionof 3-aminopropyl cinnatate hydrochloride (Tokyo Chemical Industry Co.,Ltd.) were added to a solution of sodium hyaluronate (1.06 g, 2.7mmol/disaccharide unit, weight-average molecular weight 900,000; derivedfrom cock's comb, Seikagaku Corporation) in water (115 mL)/dioxane (144mL). The mixture was stirred for 3 hours and a 750 mg/10 mL aqueoussolution of sodium hydrogen carbonate (Japanese Pharmacopoeia) wasadded. After stirring further 2 h 30 min., the reaction was quenchedwith acetic acid (214 mg) and sodium chloride (1.0 g). Ethanol (300 mL)was added and the resulting precipitation was filtered off and washedtwice successively by 80% ethanol, 95% ethanol. The solid was dried invacuo at 40° C. over night to afford white solid (1.06 g)(hereinafter inExamples, “cinnamate derivative-introduced sodium hyaluronate” isabbreviated as “cinnamate derivative-introduced HA”). The introductionratio of the cinnamate derivative was 16%. The introduction ratio of thecinnamate derivative was calculated based on the amount of cinnamate byan absorbance measurement method (wavelength: 269 nm) and the amount ofhyaluronate by a carbazole sulfate method.

(1-2) Preparation of Cinnamate Derivative-Introduced HA Solution

Saline was added to 86 mg of cinnamate derivative-introduced HA obtainedin the above (1-1) to give a total amount of 15.45 ml, then the solutionwas shaken over night with a shaker until uniformly dissolved. The 0.5%by weight solution of cinnamate derivative-introduced HA (in drying loss10%) was obtained. Likewise, the 0.3% by weight and 0.1% by weightsolutions of cinnamate derivative-introduced HA were obtained.

Example 2 (2-1) Preparation of Cinnamate Derivative-Introduced HA

A 75 mg/5 mL aqueous solution. of HOSu, a 62 mg/5 mL WFI solution ofEDCI.HCl, and a 92 mg/5 mL WFI solution of 6-aminohexyl cinnamatehydrochloride (Tokyo Chemical Industry Co., Ltd.) were added to asolution of sodium hyaluronate (1.0 g, 2.5 mmol/disaccharide unit,weight-average molecular weight 1,500,000; derived from cock's comb,Seikagaku Corporation) in water for injection (hereinafter referred toas WFI) (150 mL)/dioxane (75 mL). The mixture was stirred for 4 hoursand sodium chloride (1.0 g) was added. Ethanol (500 mL) was added andthe resulting precipitation was filtered off and washed twicesuccessively by 80% ethanol, ethanol. The solid was dried in vacuo at40° C. to afford white solid (1.1 g). The introduction ratio of thecinnamate derivative was 2.7%.

(2-2). Preparation of Crosslinked Cinnamate Derivative-Introduced HA

The above cinnamate derivative-introduced HA (12.5 g) was dissolved inphosphate buffered saline (concentration of phosphate: 1.5 mM,hereinafter abbreviated as “PBS”) to prepare 2.5% solution of cinnamatederivative-introduced HA (500 mL). The 2.5% solution of cinnamatederivative-introduced HA was irradiated by 800 W high pressure mercurylamp and performed by a heat treatment in an autoclave at 121° C. for7.5 min. to yield crosslinked cinnamate derivative-introduced HA.

Further, 1 g of the above crosslinked cinnamate derivative-introduced HAwas dissolved in 11.5 ml of WFI to prepare the 0.2% by weight ofcrosslinked, cinnamate derivative-introduced HA.

Example 3 (3-1) Preparation of Cinnamate Derivative-Introduced HA

A 172 mg/5 mL aqueous solution of HOSu, a 143 mg/5 mL aqueous solutionof EDCI.HCl, and a 181 mg/5 mL aqueous solution of 3-aminopropylcinnamate hydrochloride (Tokyo Chemical Industry Co., Ltd.) were addedto a solution of sodium hyaluronate (1.0 g; 2.5 mmol/disaccharide unit,weight-average molecular weight 900,000; derived from cock's comb,Seikagaku Corporation) in water (150 mL)/dioxane (75 mL). The mixturewas stirred for 3 hours and a 750 mg/10 mL aqueous solution of sodiumhydrogen carbonate (Japanese Pharmacopoeia) was added. After stirringfurther 2 h 30 min., the reaction was quenched with acetic acid (214 mg)and sodium chloride (1.0 g). Ethanol (300 mL) was added and theresulting precipitation was filtered off and washed twice successivelyby 80% ethanol, 95% ethanol. The solid was dried in vacuo at 40° C. toafford white solid (1.0 g) as cinnamate derivative-introduced HA. Theintroduction ratio of the cinnamate derivative was 10.1%.

(3-2). Preparation of Fluorochrome-Labeled CinnamateDerivative-Introduced HA

A 3.0 mmol/mL aqueous solution of HOSu, a 1.5 mmol/mL aqueous solutionof EDCI.HCl and a 1.5 mmol/mL aqueous solution of 4-aminofluorescein(Tokyo Chemical Industry Co., Ltd.) were added to a solution ofcinnamate derivative-introduced HA obtained in the above (3-1)(1.00 g,2.5 mmol/disaccharide unit) in water (150 mL)/dioxane (75 mL). Themixture was stirred one day and a 500 mg/10 mL aqueous solution ofsodium hydrogen carbonate (Japanese Pharmacopoeia) was added. Afterstirring further 4 h 30 min., the reaction was quenched with acetic acid(2 mL) and sodium chloride (6.0 g). Ethanol (500 mL) was added and theresulting precipitation was filtered off and washed four times by 80%ethanol, twice by ethanol. The solid was dried in vacuo over night toafford fluorochrome-labeled solid (782 mg) The introduction ratio of thefluorescence was 0.60%.

Comparative Example 1 Preparation of Fluorochrome-Labeled HA

A 3.0 mmol/mL aqueous solution of HOSu, a 1.5 mmol/mL aqueous solutionof EDCI.HCl and a 1.5 mmol/mL, aqueous solution of 4-aminofluorescein(Tokyo Chemical Industry Co., Ltd.) were added to a solution of sodiumhyaluronate(1.00 g, 2.5 mmol/disaccharide weight-average molecularweight 900,000; derived from cock's comb, Seikagaku Corporation) inwater (150 mL)/dioxane (75 mL). The mixture was stirred one day and a500 mg/10 mL aqueous solution of sodium hydrogen carbonate (JapanesePharmacopoeia) was added. After stirring further 4 h 30 min., thereaction was quenched with acetic acid (2 mL) and sodium chloride (6.0g). Ethanol (500 mL) was added and the resulting precipitation wasfiltered off and washed four times by 80% ethanol, twice by ethanol. Thesolid was dried in vacuo, over night to afford fluorochrome-labeledsolid (830 mg) The introduction ratio of the fluorescence was 0.32%.

Example 4 Measurement of Ultraviolet Ray Transmittance of CinnamateDerivative-Introduced HA Solution

The 0.1% by weight aqueous solution of cinnamate derivative-introducedHA obtained in the above (1-1) was prepared, and the ultraviolet raytransmittance was measured by an spectrometer (UV-1600, ShimadzuCorporation).

A spectrum which indicates the transmittance is shown in FIG. 1, and thetransmittance (%) at various wavelengths is shown in Table 1. As aresult, 100% of the transmittance was shown at the wavelengths of 340 nmor more, but the transmittance at the wavelengths of approximately 320nm or less was 20% or less which was extremely low, and it wasdemonstrated that this solution effectively blocks the transmission ofthe ultraviolet ray.

In FIG. 1, scales are shown with 65 nm intervals on a horizontal axisand with 20% intervals on a vertical axis.

TABLE 1 λ T(%) 234 19.9 340 106.2 380 101.8 450 98.5

In the table, λ and T represent the wavelength and the transmittance(%), respectively.

Example 5

The effect of the cinnamate derivative-introduced HA on the healing of,rabbit corneal epithelium.

The effect of the cinnamate derivative-introduced HA prepared in Example1 on the healing of rabbit corneal epithelium with surgical removal (Thesurgical Model).

(5-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour and 4 hours after the corneal epithelia were peeled, 150 ul ofsaline as the control substance was administered in the left eye, and150 ul of 0.5% by weight cinnamate derivative-introduced HA solutionprepared in the above Example (1-2) as the subject substance wasadministered in the right eye. On one day and 2 days after the peeling,a total of 4 times with 3 hour intervals, and at 3 days after thepeeling, with 3 hours interval, the same administration as above wasperformed. In the administration, 1 ml injection syringes were used. Sixmodel rabbits for the corneal epithelial layer disorder described in theabove 1) were used as administration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 3 hours after the finaladministration at 3 days after the peeling. When photographed, a focallength was made constant to make a magnification of photographsconstant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using an imageanalyzer. A value obtained by subtracting the area of the peeled site 3hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after peeling the corneal epitheliawas performed was rendered as “healed area.”

(5-2) Study Results

The results of a healed area percentage in each individual are shown inFIG. 2, and the results of the healed area percentage and a healed areapercentage ratio in each individual are shown in Table 2. The healedarea percentage and the healed area percentage ratio were calculated asfollows.

Healed area percentage (%)=(Healed area/Peeled area)×100

Healed area percentage ratio=(Healed area percentage in right eye/Healedarea percentage in left eye)×100

TABLE 2 Healed area percentage (%) right eye (administration of left eyeHealed area Specimen cinnamate derivative- (administration percentageNumber introduced HA) of saline) ratio 1 76.90 66.59 115.47 2 69.4485.90 80.85 3 75.23 61.71 121.91 4 75.54 56.79 133.01 5 68.41 64.53106.01 6 83.76 66.92 125.16 Mean 74.88 67.07 113.73 Standard 5.57 9.95 —deviation

For each individual of individual numbers 1 to 6, the left column showsthe healed area percentage in the right eye (administration of cinnamatederivative-introduced HA), and the right column shows the healed areapercentage in the left eye (administration of saline). In FIG. 2 andTable 2, the apparent effect to facilitate the healing of the cornealepithelial layer disorder was observed in 5 of the administered 6individuals.

Example 6 The Effect of the 0.5% Cinnamate Derivative-Introduced HA onthe Healing of Rabbit Corneal Epithelium

The effect of the cinnamate derivative-introduced HA prepared in Example1 on the healing of rabbit corneal epithelium with surgical removal (Thesurgical Model).

(6-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketatine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour and 4 hours after the corneal epithelia were peeled, 150 ul ofsaline as the control substance was administered in the left eye, and150 ul of 0.5% by weight cinnamate derivative-introduced HA solutionprepared in the above Example (1-2) as the subject substance wasadministered in the right eye. On one day and 2 days after the peeling atotal of 4 times with 3 hour intervals, and at 3 days after the peeling,with 3 hour intervals, the same administration as above was performed.In the administration, the 1 ml injection syringes were used. Fourteenmodel rabbits for the corneal epithelial layer disorder described in theabove 1) were used as the administration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 3 hours after the secondadministration at 1 to 3 days after the peeling. When, photographed, thefocal length was made constant to make the magnification of photographsconstant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value obtained by subtracting the area of the peeled site3 hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after the corneal epithelia werepeeled was rendered as “healed area.”

(6-2) Study Results

The results of the healed area in each individual are shown in FIG. 3,and the results of a healing rate in each individual are shown in FIG.4. The healed area and the 20 healing rate were calculated as follows:

Healed area=Area after peeling*−Area at each time point (after 1 to 3days)

* Herein after, “area after peeling” means ‘just before theadministration of the subject substance one hour after the cornea waspeeled’ in calculation of healed area.

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 3 and 4, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.5%by weight cinnamate derivative-introduced HA solution, compared with thehealed area of the corneal epithelia in the control eyes. And it wasalso observed that the healing rate was significantly enhanced in theeyes administered with 0.5% by weight cinnamate derivative-introduced HAsolution.

Example 7 The Effect of the 0.3% Cinnamate Derivative-Introduced HA onthe Healing of Rabbit Corneal Epithelium

The effect of the cinnamate derivative-introduced HA prepared in Example1 on the Migration of rabbit corneal epithelium with surgical removal(The surgical Model).

(7-1) Methods 1) Surgical Removal of Corneal Epithelium (the Surgical 10Model)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylaliine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour and 4 hours after peeling the corneal epithelia, 150 ul ofsaline as the control substance was administered in the left eye, and150 41 of 0.3% by weight cinnamate derivative-introduced HA solutionprepared in the above Example (1-2) as the subject substance was.administered in the right eye. At one day and 2 days after the peeling,a total of 4 times with 3 hour intervals, and at 3 days after thepeeling, with 3 hour intervals, the same administration as above wasperformed. In the administration, the 1 ml injection syringes were used.Fourteen model rabbits for the corneal epithelial layer disorderdescribed in the above 1) were used as the administration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 3 hours after the secondadministration at 1 to 3 days after the peeling. When photographed, thefocal 5 length was made constant to make the magnification ofphotographs constant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value obtained by subtracting the area of the peeled site3 hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after the corneal epithelia werepeeled was rendered as “healed area.”

(7-2) Study Results

The results of the healed area in each individual are shown in FIG. 5,and the results, of a healing rate in each individual are shown in FIG.6. The healed area and the healing rate were calculated as follows.

Healed area=Area after peeling−Area at each time point (after 1 to 3days)

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 5 and 6, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.3%by weight cinnamate derivative-introduced HA solution at all time pointsof days 1 to 3, compared with the healed area of the corneal epitheliain the control eyes. And it was also observed that the healing rate wassignificantly enhanced in the eyes administered with 0.3% by weightcinnamate derivative-introduced HA solution.

Example 8 The Effect of the 0.1% Cinnamate Derivative-Introduced HA onthe Healing of Rabbit Corneal Epithelium (0.1% by Weight CinnamateDerivative-Introduced HA Aqueous Solution and 0.1% by Weight HA AqueousSolution, 4 Times of Eye Drops Per Day)

The effect of the cinnamate derivative-introduced HA prepared in Example1 on the healing of rabbit corneal epithelium with surgical removal (Thesurgical Model).

(8-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour and 4 hours after peeling the corneal. epithelia, 150 ul of anaqueous solution of 0.1% by. weight HA with a weight average molecularweight 600,000 to 1,200,000 as the control substance was administered inthe left eye, and 150 ul of 0.1% by weight cinnamatederivative-introduced HA solution prepared in the above Example (1-2) asthe subject substance was administered in the right eye. At one day and2 days after the peeling, a total of 4 times with 3 hour intervals, andat 3 days after the peeling, with 3 hour intervals, the sameadministration as above was performed. In the administration, the 1 mlinjection syringes were used. Eight model rabbits for the cornealepithelial layer disorder described in the above 1) were used as theadministration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea, was peeled and 3 hours after the secondadministration at 1 to 3 days after the peeling. When photographed, thefocal length was made constant to make the magnification of photographsconstant′.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value 15 obtained by subtracting the area of the peeledsite, 3 hours after the final administration at 3 days after the peelingfrom the area (peeled area) of the peeled site just before theadministration of the subject substance one hour after the cornealepithelia were peeled was rendered as “healed area.”

(8-2) Study Results

The results of the healed area in each individual are shown in FIG. 7,and the results of the healed area and the healing rate in eachindividual are shown in FIG. 8. The healed area and the healing ratewere calculated as follows.

Healed area=Area after peeling−Area at each time point (after 1 to 3days)

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 7 and 8, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.1%by weight cinnamate derivative-introduced HA solution, compared with thehealed area of the corneal epithelia in the control eyes administeredwith the 0.1% by weight HA aqueous solution. And it was also observedthat the healing rated was significantly enhanced in the eyesadministered with 0.1% by weight cinnamate derivative-introduced HAsolution.

Example 9 The Effect of the 0.1% Cinnamate Derivative-Introduced HA onthe Migration of Rabbit Corneal Epithelium (0.1% by Weight CinnamateDerivative-Introduce a HA Aqueous Solution and 0.1% by Weight HA AqueousSolution, One Time of Eye Drops Per Day)

The effect of the cinnamate derivative-introduced HA prepared in Example1 on the Migration of rabbit corneal epithelium with surgical removal(The surgical Model).

(9-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour after the corneal epithelia were peeled, 150 ul of the aqueoussolution of 0.1% by weight HA with a weight average molecular weight600,000 to 1,200,000 as the control substance was administered in theleft eye, and 150 ul of 0.1% by weight cinnamate derivative-introducedHA solution prepared in the above Example (1-2) as the subject substancewas administered in the right eye. Furthermore, at one to 3 days afterthe peeling, once a day, the same administration as above was performed.In the administration, the 1 ml injection syringes were used. Eightmodel rabbits for the corneal epithelial layer disorder described in theabove 1) were used as the administration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 6 hours after the administration at1 to 3 days after the peeling. When photographed, the focal length wasmade constant to make the magnification of photographs constant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value obtained by subtracting the area of the peeled site3 hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after the corneal epithelia werepeeled was rendered as the “healed area.”

9-2) Results

The results of the healed area in each individual are shown in FIG. 9,and the results of the healing rate in each individual are shown in FIG.10. The healed area and the healing rate were calculated as follows.

Healed area=Area after peeling−Area at each time point (after 1 to 3days)

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 9 and 10, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.1%by weight cinnamate derivative-introduced HA solution at all time pointsof the days 1 to 3, compared with the healed area of the cornealepithelia in the control eyes administered with the 0.1% by weight HAaqueous solution. And it was also observed that the healing rate wassignificantly enhanced in the eyes administered with 0.1% by weightcinnamate derivative introduced HA solution.

Example 10 Staying Property at Rabbit Corneal Epithelial Peeling SiteUsing Fluorescence Labeled Cinnamate Derivative-Introduced HA

The effect of the fluorescence labeled cinnamate derivative-introducedHA prepared in Example and the fluorescence labeled HA prepared inComparative Example 1 on the residual property of rabbit cornealepithelium with surgical removal (The surgical Model).

(101-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour after the corneal epithelia were peeled, 150 ul of the aqueoussolution of 0.3% by weight fluorescence labeled HA prepared in the aboveComparative Example 1 as the control substance was administered in theleft eye, and 150 ul of the aqueous solution of 0.3% by weightfluorescence labeled cinnamate derivative-introduced HA prepared in theabove Example 3 as the subject substance was administered in the righteye. In the administration, the 1 ml injection syringes were used. Eightmodel rabbits for the corneal epithelial layer disorder described in theabove 1) were used as the administration subjects.

3) Removal of Corneal Epithelia and Production of Frozen Blocks

Two rabbits were given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine per rabbit, and eyeballs wereremoved 30 minutes, one hour, one and a half hours and 2 hours and 30minutes after the administration of the subject substance and thecontrol substance. A pore was opened between the cornea and sclera inthe removed eyeball using a surgical knife, and only the cornea wastaken out using the microscissors. The removed cornea was placed on abiological sample slicing plate (supplied from Nisshin EM Corporation,Cat No. 428), and the portion to be observed was cut out using asingle-edged razor blade stainless steel (GEM® STAINLESS STEEL UNCOATED,Nisshin EM Corporation, Cat No. 429). The cut out portion was immersedin O.C.T. compound (Tissue-Te) (R)4583, Lot. 1178), then embedded in acryostat tray (supplied by Murazumi Co., Ltd., Cat. No. 31) filled withthe O.C.T. compound so that the portion to be observed was at a bottom,and rapidly frozen using liquid nitrogen in foam polystyrene to make anunfixed frozen block.

4) Production of Frozen Sections

Subsequently, the frozen block was removed from the cryostat tray, andattached on a sample table using the O.C.T. compound. The sample tableand a disposable microtome blade (supplied by Leica Microsystems Japan,Model 818, Lot. No. 913212) were set in a high performance frozenmicrotome for research, and the block was sliced under the condition ofa frozen chamber temperature (CT) at −20° C. and sample side temperature(OT) at −16° C. to make sections with a thickness of 5 uM using silanecoating slide glasses (supplied by Muto Pure Chemicals Co., Ltd., StarFrost Slide Glass, Cat. No. 5116).

5) Methods of Observation and Photographing

The frozen section was set in an incident-light fluorescence microscope(Olympus Corporation, BH2-RFC), FA images and autofluorescent imageswere observed at IB cube BH2-DMIB, excitation wavelength: 495 nm,absorption wavelength: 460 nm) and U cube (BH2-DMU, broad band Uexcitation, absorption wavelength: 435 nm), respectively. The FA imageand the autofluorescent image were photographed using a cooled highsensitivity CCD camera (Keyence Corporation, VB-6010) under thecondition of exposure time for one second and ISO sensitivity of 200.

(10-2) Study Results

Photographs of the sampled cornea were shown in FIGS. 11 and 12. FromFIGS. 11 and 12, it was identified by color development of thefluorescence label that the aqueous solution of 0.3% by weightfluorescence labeled HA which was the control substance stayed until 30minutes after. the administration but did not stay after one hour.Meanwhile, although the fluorescent color development of the aqueoussolution of 0.3% by weight fluorescence labeled cinnamatederivative-introduced HA which was the subject substance was weakenedwith the elapse of time, the color development was observed at all timepoints from 30 minutes to 2 hours and 30 minutes after theadministration, thereby the high staying performance was confirmed.

Example 11 Effect of the 0.3% Cinnamate Derivative-Introduced HA onRabbit Eyes after Exposure to Ultraviolet Light

The protective effect on the cinnamate derivative introduced HA preparedin Example 1 on the rabbit with corneal superficial disorder.

(11-1) Study Procedure

1) Anesthesia and Eyelid Opening in Rabbit

Introduced anesthesia by intravenously injecting 5 mg/kg of ketimine and2 mg/kg of xylazine and maintained anesthesia by inhalation ofisoflurane were given to the rabbit. Subsequently, the eyelid was alwaysopened using an eyelid retractor for kids.

2) Administration of Subject Substance and Control Substance

In the condition where the eye was opened, 150 ul of an aqueous solutionof 0.3% by weight HA with a weight average molecular weight 600,000 to1,200,000 as the control substance was administered in the right eye,and 150 1.11 of 0.3% by weight cinnamate derivative-introduced HAprepared in the above Example (1-2) as the subject substance wasadministered in the left eye. In the administration, the 1 Ml injectionsyringes were used. One rabbit described in the above 1) was used as theadministration subject.

3) Irradiation of Ultraviolet Rays to Rabbit's Cornea

Ultraviolet rays were irradiated to both eyes from a distance ofapproximately 10 cm apart from the rabbit eyeball using a 15 kWgermicidal lamp. The irradiation was performed for 3 hours.

4) Photographing of Ultraviolet Rays Irradiated Site

The eyeball was stained with 0.2% sodium fluorescein under thecontinuous anesthesia of the rabbit, and photographed under violetlight. When photographed, the focal length was made constant to make themagnification of photographs constant.

(11-2) Study Results

Photographs after the irradiation of the ultraviolet rays were shown inFIG. 13. From FIG. 13, in the eyeball irradiated with the ultravioletrays after the administration of the control substance, the disorderedsite stained with 0.2% sodium fluorescein was apparent.

Meanwhile, in the eyeball irradiated with the ultraviolet rays after theadministration of the subject substance, the disordered site stainedwith 0.2% sodium fluorescein clearly smaller than that of the controlsubstance, and the corneal disorder caused by the ultraviolet ray wasprevented.

Example 12 Moisturizing Effect Using Removed Cornea

Using the removed cornea of the rabbit, the moisturizing performance ofthe cinnamate derivative-introduced HA prepared in Example 1 wasvalidated.

(12-1) Methods 1) Removal of Cornea

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, and the eyeball was removed.A pore was opened between the cornea and the sclera in the removedeyeball using a surgical knife, and only the cornea was taken out usingthe microscissors.

2) Drying Treatment of Cornea

The drying treatment was performed by placing the removed cornea on aparaffin block with a False-tooth stability material and providing coldair from the distance of approximately 1 m from the cornea using a dryerfor 5 minutes.

3) Administration of Test Substances (Subject Substance, ControlSubstance and Negative Control Substance)

After the completion of the drying treatment, 100 ul of saline as thenegative control substance, the aqueous solution of 0.3% by weight HAwith a weight average molecular weight of 600,000 to 1,200,000 as thecontrol substance or the aqueous solution of 0.5% by weight cinnamatederivative-introduced HA prepared in the above Example (1-2) as thesubject substance was administered to two corneas. In theadministration, the 1 ml injection syringes were used. Three rabbits (6corneas) described in the above 1) were used as the administrationsubjects.

4) Measurement of Water Evaporation Amount

The water evaporation amount was measured using a water evaporationamount measurement apparatus (AS-TW2, ASAHIBIOMED) before theadministration of the test substance, after the drying treatment, afterthe administration of the test substance and until 40 minutes with 10minute intervals after the administration of the test substance.

(12-2) Study Results

The results of measuring the water evaporation amount were shown in FIG.14. From FIG. 14, the water evaporation amount was slightly higher inthe HA aqueous solution which was the control substance than in thesaline which was the negative control whereas the saline became thevalue close to 0 after 40 minutes. On the other hand, the waterevaporation amount after the administration of the subject substancekept the high value even when 40 minutes passed over, thereby the clearmoisturizing performance of the subject substance was confirmed.

Example 13 (13-1) Methods 1) Removal of Corneal Epithelia

The rabbit was euthanized, and after removing the eyeball, an entirecorneal layer was removed by incising along the sclera. The removedcornea was preserved in saline, and the corneal epithelia was fixed byplacing it on the paraffin block and the False-tooth stability materialjust before the measurement (hereinafter described as “the cornea to bemeasured”).

2) Water Evaporation in Corneal Epithelia

The cornea to be measured was given cold air by the dryer from thedistance of 30 cm for 5 minutes, and left standing at a room temperaturefor one hour.

3) Administration of Subject Substance

After the water is evaporated, two drops (approximately 100 ul) ofsaline as the negative control substance, the aqueous solution of 0.3%by weight HA with a weight average molecular weight of 600,000 to1,200,000 as the control substance or the aqueous solution of 0.5% byweight cinnamate derivative-introduced HA prepared in the above Example(1-2) as the subject substance was administered by the 1 ml syringe.

4) Measurement of Water Evaporation Amounts

The amount perceived as an unperceived evaporation amount (releasedwater amount per m₂ per hour) was directly measured as the waterevaporation amount from the cornea to be measured using the waterevaporation amount measurement apparatus (AS-TW2).

(13-2) Study Results

The results of measuring the water evaporation amount were shown in FIG.15. From FIG. 15, the water evaporation amount was slightly higher inthe HA aqueous solution which was the control substance than in thesaline which was the negative control whereas the saline exhibited thevalue close to 0 after 40 minutes. On the other hand, the waterevaporation amount of the administered subject substance kept the highvalue even when 40 minutes had passed over, thereby it was confirmedthat the subject substance has a more persistent water retentionproperty on the cornea compared with saline and the HA aqueous solution.

Example 14 Validation of Healing Effect of Crosslinked CinnamateDerivative-Introduced HA

Using a model hamster for xerostomia, the healing effect of thecrosslinked cinnamate derivative-introduced HA prepared in Example 2 onthe xerostomia was validated.

(14-1) Test Method 1) Production of Model Hamster for Xerostomia

An inside of the oral cavity of a male Syrian hamster was exposed byinserting a test tube with a diameter of approximately 10 mm in the oralcavity close to a buccal side and reversing it under the anesthesia withNembutal. By giving hot air for approximately 20 seconds, andsubsequently giving cold air for 2 minutes and 40 seconds to theexposed, inside of the oral cavity using a dryer, the model hamster forthe xerostomia was obtained. The inside of the oral cavity wascontinuously exposed until the measurement was completed.

2) Administration of Subject Substance and Control Substance

Immediately after making the xerostomia model, 100 ul of (A) PBS, (B)0.2% by weight HA solution (supplied by Seikagaku Corporation, weightaverage molecular weight: 1,500,000) or (C) 0.2% by weight crosslinkedcinnamate derivative-introduced HA solution was administered by applyingto the inside of the oral cavity using a microsyringe.

Hereinafter, the group administered with (A), the 25 group administeredwith (B) and the group administered with (C) are referred to as PBSgroup, HA group and crosslinked cinnamate derivative-introduced HAgroup, respectively. For the administration classification(administration group composition), seven hamsters were used for each ofthe PBS group, the HA group and the crosslinked cinnamatederivative-introduced HA group.

3) Calculation of Water Evaporation Amount Ratio

The water evaporation amount in the inside of the oral cavity in thexerostomia model hamster was measured using the water evaporation system(Asahibiomed), and the water evaporation amount ratio was calculatedwhen the measurement value immediately after making the xerostomia modelhamster was 1. The higher this water evaporation amount ratio is, themore moisturized condition is maintained (degree of oral cavity drynessis low). The measurement was performed immediately after, making theerostomia model hamster, immediately after the administration, 10minutes and 20 minutes after the administration.

(14-2) Study Results

The results of measuring the water evaporation amount are shown in FIG.16. In the figure, circled numbers 1, 2, 3 and 4 on the horizontal axisrepresent data immediately after making the xerostomia model hamster,immediately after the administration, 10 minutes and 20 minutes afterthe administration, respectively. P represents a significance level.Immediately after the administration, all of administration groupsexhibited the water evaporation amount ratio of 3.3 to 4.5. The waterevaporation amount ratio 10 minutes after the administration was 0.9 onaverage in the PBS group and 2.3 on average in the HA group. On theother hand, it was 3.2 on average in the crosslinked cinnamatederivative-introduced HA group, thus the higher water evaporation amountratio than the PBS group and the HA group was shown. Further, the waterevaporation amount ratio 20 minutes after the administration was 0.9 onaverage in the PBS group and 1.3 on average in the HA group. On theother hand, it was 3.2 on average in the crosslinked cinnamatederivative-introduced HA group, thus the extremely higher waterevaporation amount ratio compared with the PBS group and the HA groupwas indicated. Furthermore, as is apparent from FIG. 16, the waterevaporation amount ratio in the crosslinked cinnamatederivative-introduced HA group was very stable regardless of the elapseof time. This indicated that the crosslinked cinnamatederivative-introduced HA stayed at-the administered site for a longperiod of time and exerted a highly persistent effect.

From the above results, GAG into which the hydrophobic group isintroduced via the binding chain including the cinnamatederivative-introduced HA and the crosslinked cinnamatederivative-introduced HA has been shown to be suitable for theapplication to the mucosa and capable of effectively treating thedisorder in the mucosal epithelial layer by being applied to the mucosa.It has been also shown that the effect of the treatment is highlypersistent.

Since no adverse effect due to the administration of the cinnamatederivative-introduced HA and the crosslinked cinnamatederivative-introduced HA was observed in any of the above animalstudies, the safety of the agent for applying to mucosa of the presentinvention can be sufficiently estimated.

Example 15 (15-1) Preparation of Octylamine-Introduced SodiumHyaluronate

A 25.8 mg/2 mL solution (ethanol:0.1MHC1=I:1) of octylamine, 2 mL of 0.1M solution (ethanol:water=1:1) of DMT-MM (Wako Pure Chemical Industries,Ltd.) were added to a solution of sodium hyaluronate (502 mg, 1.25mmol/disaccharide unit, weight-average molecular weight 900,000) inwater (50 mL)/ethanol (50 mL). The mixture was stirred over night and a376 mg/5 mL aqueous solution of sodium hydrogen carbonate (JapanesePharmacopoeia) was added. After stirring further 5 hours, the reactionwas quenched with acetic acid (107 mg) and sodium chloride (522 mg).Ethanol (250 mL) was added and the resulting precipitation was filteredoff and washed twice successively by 80% ethanol, ethanol. The solid wasdried in vacuo to afford white solid (475 mg). The introduction ratio ofoctylamine was 12.6% by HPLC.

(15-2) Preparation of Hexadecylamine-Introduced Sodium Hyaluronate

A 30 mg/3 mL solution (ethanol:0.1MHC1=1:1) of octylamine, 1.25 mL of0.1 M solution (ethanol:water=1:1) of DMT-MM (Wako Pure ChemicalIndustries, Ltd.) were added to a solution of sodium hyaluronate (501mg, 1.25 mmol/disaccharide unit, weight-average molecular weight900,000) in water (50 mL)/ethanol (50 mL). The mixture was stirred overnight and a 381 .mg/5 mL aqueous solution of sodium hydrogen carbonate(Japanese Pharmacopoeia) was added. After stirring further 5 hours, thereaction was quenched with acetic acid (107 mg) and sodium chloride (497mg): Ethanol (250 ML) was added and the resulting precipitation wasfiltered off and washed twice successively by 80% ethanol, ethanol. Thesolid was dried in vacuo to afford white solid (497 mg). Theintroduction ratio of hexadecylamine was 12% by HPLC.

(15-3) Preparation of Sample Solution

64 mg of compound obtained in the above (15-1) was added to 5 mMphosphate buffer saline to give a total amount of 59 ml, then thesolution was shaken over night with a shaker. The 0.1% by weightsolution of compound prepared in the above (15-1) was obtained.

Likewise, 0.1% by weight solution of compound prepared in the above(15-2) was obtained.

Example 16 The Effect of the 0.1% Cinnamate Derivative-Introduced HA onthe Healing of Rabbit Corneal Epithelium

The effect of the cinnamate derivative-introduced. HA prepared inExample 1 on the healing of rabbit corneal epithelium with surgicalremoval (The surgical Model).

(16-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One hour and 4 hours after peeling the corneal epithelia, 150 ul ofsaline as the control substance was administered in the left eye, and150 ul of 0.1% by weight cinnamate derivative introduced HA solutionprepared in the above Example (1-2) as the subject substance wasadministered in the right eye. At one day and 2 days after the peeling,a total of 4 times with 3 hour intervals, and at 3 days after thepeeling, with 3 hour intervals, the same administration as above wasperformed. In the administration, the 1 ml injection syringes were used.Fourteen model rabbits for the corneal epithelial layer disorderdescribed in the above 1) were used as the administration subjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 3 hours after the secondadministration at 1 to 3 days after the peeling. When photographed, thefocal length was made constant to make the magnification of photographsconstant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value obtained by subtracting the area of the peeled site3 hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after the corneal epithelia werepeeled was rendered as “healed area.”

(16-2) Study Results

The results of the healed area in each individual are shown in FIG. 17,and the results of a healing rate in each individual are shown in FIG.18. The healed area and the healing rate were calculated as follows.

Healed area=Area after peeling−Area at each time point (after 1 to 3days)

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 17 and 18, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.1%by weight cinnamate derivative-introduced HA solution at all time pointsof days 1 to 3, compared with the healed area of the corneal epitheliain the control eyes. And it was also observed that the healing rate wassignificantly enhanced in the eyes administered with 0.1% by weightcinnamate derivative-introduced HA solution.

Example 17 The Effect of the 0.1% Octylamine-Introduced HA andHexadecylamine-Introduced HA on the Healing of Rabbit Corneal Epithelium

The effect of the octylamine-introduced HA and hexadecylamine-introducedHA prepared in Example 15 on the healing of rabbit corneal epitheliumwith surgical removal (The surgical Model).

(17-1) Methods 1) Surgical Removal of Corneal Epithelium (the SurgicalModel)

The corneal epithelium of the central region was removed by using atrephine (8 mm I.D), a 23 G needle and microscissors after anesthesiawith an intravenous injection of 5 mg/kg of ketamine and 2 mg/kg ofxylazine and topical administration of 0.4% oxbuprocaine hydrochloride.

2) Topical Administration

One, hour and 4 hours after peeling the corneal epithelia, 150 ul ofsaline as the control substance was administered in the left eye; and150 ul, of 0.1% octylamine-introduced HA and hexadecylamine-introducedHA solution prepared in the above Example (15-2) as the subjectsubstance was administered in the right eye. At one day and 2 days afterthe peeling, a total of 4 times with 3 hour intervals, and at 3 daysafter the peeling, with 3 hour intervals, the same administration asabove was performed. In the administration, the 1 ml injection syringeswere used. Eight model each rabbits for the corneal epithelial layerdisorder described in the above 1) were used as the administrationsubjects.

3) Photographing of Corneal Epithelial Defective Region

The rabbit was given general anesthesia by intravenously injecting 5mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the cornealepithelial loss site was stained with 0.2% sodium fluorescein dissolvedin PBS, and photographed under ultra-violet light. The photographing wasperformed just before the administration of the subject substance onehour after the cornea was peeled and 3 hours after the secondadministration at 1 to 3 days after the peeling. When photographed, thefocal length was made constant to make the magnification of photographsconstant.

4) Measurement of Corneal Epithelial Defective Region

The area of the corneal epithelial defective region stained with sodiumfluorescein was measured on the printed photograph using the imageanalyzer. The value obtained by Subtracting the area of the peeled site3 hours after the final administration at 3 days after the peeling fromthe area (peeled area) of the peeled site just before the administrationof the subject substance one hour after the corneal epithelia werepeeled was rendered as “healed area.”

(17-2.) Study Results

The results of the healed area in each individual are shown in FIG. 19,and the results of a healing rate in each individual are shown in FIG.20. In FIG. 19, C8-L(a: control), C8-R(b), C16-L(c: control), C16-R(d)represents left eye to which saline was administrated as a control forC8-R, right eye to which 0.1% octylamine-introduced HA solution wasadministrated, left eye to which saline was administrated as a controlfor C16-R, right eye to which 0.1% hexadecylamine-introduced HA solutionwas administrated, respectively. In FIG. 20, C8 represents results ofthe above study using octylamine, and C16 represents results of theabove study using hexadecylamine. The healed area and the healing ratewere calculated as follows.

Healed area=Area after peeling−Area at each time point (after 1 to 3days)

Healing rate=Mean of healed areas at respective time points (after 1 to3 days)

In FIGS. 19 and 20, it was observed that the healed area of the cornealepithelia was significantly increased in the eyes administered with 0.1%octylamine-introduced HA and hexadecylamine-introduced HA solution atall time points of days 1 to 3, compared with the healed area of thecorneal epithelia in the control eyes. And it was also observed that thehealing rate was significantly enhanced in the eyes administered with0.1% octylamine-introduced HA or hexadecylamine-introduced HA solution.

1-18. (canceled)
 19. A method of producing a glycosaminoglycan intowhich a hydrophobic group is introduced via a binding chain, comprising:reacting a glycosaminoglycan with a hydrophobic compound in which saidhydrophobic group is bound to a functional group selected from the groupconsisting of hydroxyl, carboxyl, amino, and sulfonate group to form abond selected from the group consisting of an ether bond, a carboxylateester bond, a sulfate ester bond, carboxylic acid amide bond, or asulfonate amide bond together with a carboxyl, hydroxyl or sulfonategroup in said glycosaminoglycan.
 20. A method according to claim 19,wherein a hydrophobic compound, in which said functional group is anamino group, is reacted with a glycosaminoglycan, which contains acarboxyl group, to obtain a carboxylic acid amide bond.
 21. A methodaccording to claim 19, wherein a hydrophobic compound, in which saidfunctional group is a hydroxyl group, is reacted with aglycosaminoglycan, which contains a carboxyl group, to obtain acarboxylate ester bond.
 22. A method according to claim 19, wherein ahydrophobic compound, in which said functional group is a carboxylgroup, is reacted with a glycosaminoglycan, which contains a hydroxylgroup, to obtain a carboxylate ester bond.
 23. A method according toclaim 19, wherein a hydrophobic compound, in which said functional groupis a hydroxyl group, is reacted with a glycosaminoglycan, which containsa hydroxyl group, to obtain an ether bond.
 24. A method according toclaim 19, wherein a hydrophobic compound, in which said functional groupis a hydroxyl group, is reacted with a glycosaminoglycan, which containsa sulfonate group, to obtain a sulfonate ester bond.
 25. A methodaccording to claim 19, wherein a hydrophobic compound, in which saidfunctional group is a sulfonate group, is reacted with aglycosaminoglycan, which contains a hydroxyl group, to obtain asulfonate ester bond.
 26. A method according to claim 19, wherein saidglycosaminoglycan is selected from the group consisting of hyaluronicacid, chondroitin, chondroitin sulfate, heparin, heparan sulfate,dermatan sulfate and keratan sulfate, and a salt thereof.
 27. A methodaccording to claim 19, wherein said glycosaminoglycan is sodiumhyaluronate.
 28. A method according to claim 19, wherein saidhydrophobic group is selected from the group consisting of an alkylgroup having 2 to 18 carbon atoms, an alkenyl group having 2 to 18carbon atoms, an alkynyl group having 2 to 18 carbon atoms, an arylgroup, a heteroaryl group, an arylalkyl group, an arylalkenyl group, anarylalkynyl group, and an amino acid group.
 29. A method according toclaim 19, wherein said hydrophobic group is an arylalkenyl group.
 30. Amethod according to claim 19, wherein said hydrophobic group is aphenylethenyl group.